Electron transfer in photosystem II

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Abstract. The picture presently emerging from studies on the mechanism of photosystem. II electron transport is discussed. The reactions involved in excitation ...

Photosynthesis Research 6, 97-112 (1985) © 1985 Martinus Nifhoff/Dr W. Junk Publishers, Dordrecht. Printed in the Netherlands.

Electron transfer in photosystem II H.J. VAN GORKOM Department of Biophysics, Huygens Laboratory of the State University, P.O. Box 9504, 2300 RA Leiden, The Netherlands (Received: June 18, 1984; accepted November 7, 1984)

Key words: Electron transport, Photosystem II, Primary reaction Abstract. The picture presently emerging from studies on the mechanism of photosystem II electron transport is discussed. The reactions involved in excitation trapping, charge separation and stabilization of the charge pair in the reaction center, followed by the reactions with the substrates, plastoquinone reduction and water oxidation, are described successively. Finally, a brief discussion on photosystem II heterogeneity is presented.

I. Introduction Photosystem II (PS II), the oxygen evolving system of photosynthesis, is usually thought of as a photoelectric device driving electrons from water to plastoquinone. The mobile plastoquinone/plastoquinol pool is used by the rest of the photosynthetic machinery as a source of electrons for reductive synthesis of cell material, and as a lipophilic hydrogen carrier for pumping protons across the membrane to drive ATP synthesis. PS II itself contributes to this latter function as well, by releasing the protons set free by water oxidation into the thylakoid lumen, and taking the protons required for plastoquinone reduction from the chloroplast stroma, at the opposite side of the membrane. Several functional parts of PS II may be distinguished, and can to some extent be correlated with its different structural subunits. The structure and function of the pigment antenna will not be discussed here. It consists of several chlorophyll-proteins, one of which also contains the redox compounds involved in the photophysical processes of excitation trapping and charge separation. In this 'reaction center protein', the chlorophyll excited state is first transformed into a pair of porphyfin radicals, the chlorophyll a cation P+ and the pheophytin a anion I-, and subsequently into a pair of plastosemiquinone radicals, the cation Z + and the anion Q-, located effectively on opposite sides of the membrane dielectric barrier, and some 106 times longer lived than the chlorophyll excited state - long enough for its use in subsequent biochemistry. The binding and oxidation of water probably takes place at a cluster of GOV-84-RP-003

98 four manganese ions, where the oxidizing equivalents produced by successive photoreactions are accumulated. The successive redox states are called So, $1, $2 and S~. Upon arrival of the fourth positive charge the cluster is reduced to So, releasinga dioxygen molecule. The manganese cluster is kept in place by an extrinsic 33 kDa polypeptide. Ca2+ and C1- are essential for its functioning and these ions in turn are kept in place by an extrinsic 24 kDa protein. The binding and reduction of plastoquinone involves an intrinsic 32 kDa polypeptide, which is suspected of having an important regulatory role. Most herbicides attack at this point. The reduction of a plastoquinone molecule requires two electrons and these are provided by two successive photoreactions of the same PS II center. In between, a plastosemiquinone anion remains tightly bound. We are still searching for the role of some compounds known to be present, the most obvious example being cytochrome bs59, and also for the compounds responsible for some known phenomena. A discussion of these open questions is beyond the aim of the present review. However, a variety of observations 'do not fit' and seem to indicate the existence of a different kind of PS II, or at least a very different PS II functioning, as will be described briefly. In the following sections, these aspects of PS II electron transport will be discussed successively with regard to the fundamental questions: what it is, what it does, and why. The discussion is unavoidably biased by the author's present views, doubts and ignorance, and more extensive documentation may be found in recent reviews, amongst others, by Velthuys [110] and Govindjee [40] on similar topics, by Parson and Ke [79] on primary reactions, including other photosystems than PS II, by Klimov and Krasnovskii [61] on the intermediary acceptor pheophytin in PS II, by Bouges-Bocquet [15] on electron transfer reactions on the oxidizing (electron donor) side of PS II, by Amesz [3] on the role of manganese in photosynthetic oxygen evolution, by Vermaas and Govindjee [115] on electron acceptors in PS II, and by Crofts and Wraight [24] on subsequent electron transport. In addition, reviews to be published in Photosynthesis Research include: the mechanism of O2 evolution (G. Renger and Govindjee [81a] ), the polypeptides of PS II (D.F. Ghanotakis and C.F. Yocum [37a] ) and Photosystem I (A.W. Rutherford and P. Heathcote [84a]). II. The primary radical pair In photosystem II, like in other photosynthetic systems, light absorption in the antenna pigments generates an electronic excitation, which is rapidly transferred from one pigment molecule to the next, and which eventually reaches the photochemically active chlorophyll, P. In PS II, P consists of chlorophyll a with a long wavelength absorption maximum near 680 nm, close to that of the bulk chlorophyll a (reviewed e.g., in [4] ). So its excitation as such does not help to trap the excitation energy. The number of chlorophyll

99 a molecules involved is the subject of a continuing, perhaps in part semantic, discussion (reviewed in [48]) and need not concern us here; the reaction center protein contains plenty of chlorophyll a molecules. The important point for the present discussion is that, upon excitation of P, a charge separation can take place, and the primary radical pair P+I- is formed. In experiments where one of these two radicals was accumulated, P+ has been identified as a chlorophyll a radical cation [48] and I-as a pheophytin a radical anion [61]. This is confirmed by the only direct observation of the radical pair reported so far [91]. In this study a PS II 'core' preparation prepared with Triton X-100 was used, in which the antenna size is about 5 times smaller than in intact PS II, and the kinetics may also have been modified by the detergent. When Q was in the reduced state, P+I- had a lifetime of 4 ns. It was accompanied by an intense delayed fluorescence, clearly slower than the main 1 ns fluorescence decay, but direct recombination to the triplet state was reported as well [60]. These results are basically similar to what is observed in reaction centers from purple bacteria [79]. In intact PS II at physiological temperatures no direct recombination of P+I- to the triplet state seems to occur when Q is in the reduced state [60, 64, 100]. The fluorescence emission then has a lifetime of about 2ns [e.g., in 43], which should be long enough for some triplet formation [47]. The absence of triplet formation may have three different causes: (1) P+I- is not formed; (2) P÷I- stays in its initial singlet configuration and does not exhibit the expected electron spin dephasing on a 2 ns timescale; and (3) P÷I- decays to the ground state more rapidly than to the triplet state. In our opinion this last possibility is unlikely, since no substantial fluorescence quenching by PS II centers in the state P I Q- seems to occur [100] and also because the observed fluorescence emission is not thermally activated [69]. The first possibility is compatible with efficient photosynthesis only if it is the presence of Q- which lifts the energy level of P÷I- to or above that of the singlet excited state, but in addition it is hard to reconcile it with the observed light-induced accumulation of the state P I-Q-at low redox potential [61 ]. Thus we favor the hypothesis that P÷I- is somehow kept in the singlet configuration and decays via reexcitation of chlorophyll only. As pointed out in ref. [105], a high enough rate constant of the back reaction P+I--+ P* may effectively prevent electron spin dephasing since the latter process initially proceeds as a quadratic function of time [47]. The back reaction would have to be much faster than the nanoseconds process of electron spin dephasing, perhaps in the order of 100 ps. If the photochemical charge separation takes in the order of 1 ps (but that is merely a plausible guess), the equilibrium constant of the charge separation, P* ~ P+I-, might about compensate the 200-fold degeneracy of the excited state hopping-around in the PS II antenna:

(200Chla)* K=l/2OO_ p, K~209 P+IIt may be concluded that not only the excitation transfer towards P, but also

100 the formation of the radical pair P+I- is primarily a mechanistic requirement and by itself does not contribute very much to increase the lifetime of the free energy supplied by an excitation.

III. Stabilization by electron transfer to Q The next step is electron transfer from I- to Q, the traditional "primary" electron acceptor in PS II [35]. Research in this area has very profitably followed the lead of research on reaction centers isolated from purple bacteria. Q, a tightly bound plastoquinone molecule, is reduced to the unprotonated, anionic semiquinone, accompanied by electrochromic bandshifts of pheophytin a (one of which is well-known as "C-550"), but not by the semiquinone ESR signat at g = 2 [104]. The ESR properties of Q- are determined largely by its interaction with a nearby non-heine iron [76]. A Q--Fe signal is observed at g = 1.82 or 1.90, depending on the pH (6 or 8, respectively) [87] and the presence of Q--Fe causes a splitting of the ESR signal of I- [59]. Low temperature ESR has become a very useful tool to study the pheophytin-quinone-iron arrangement (reviewed in ref. [84] ). The overall process, from light absorption to Q reduction, takes about 100 or about 400 ps, depending on the interpretation of the observed fluorescence lifetimes [56, 108]. The rate constant of electron transfer from I- to Q is unknown and may be substantially higher than this time suggests. It is this reaction which actually traps the excitation energy by its large exothermicity. The charge recombination P+Q- -+ P Q takes about 200#s [45], as compared to 2 ns for P+I- -~ P I in the presence of Q-. The latter reaction proceeds via reexcitation of chlorophyll and subsequent loss to the ground state, in part via fluorescence emission (see previous section). By comparison of the emission yields, it was found that this route accounts for only 3% of the decay of P+Q- [25]. Thus, if no other decay routes of P+Q-existed, it would be 3 x 106 times more stable than P+I-. The 2 ns lifetime of P+I-relates to the singlet configuration of this radical pair only, as discussed in the previous section, whereas no electron spin correlation can be assumed in the state P+Q-. In view of the threefold degeneracy of the triplet state we assume that one in four back reactions of P+Q- yields a radical pair P+I- in a spin state which allows recombination to the singlet excited state, and thus obtain an equilibrium constant of 8 x l0 s for the reaction I-Q ~ I Q-. If the state which determined the 2 ns fluorescence lifetime is largely excited chlorophyll rather than P+I- (see previous section), the equilibrium constant of I-Q ~ I Q- may be up to 200 times larger still. The-minimum value of 8 x l0 s corresponds (at 20°C) to a free energy difference between I-Q and I Q- of AG = 0.058 log K = 0.34 eV. This (minimum) value just meets the maximum value of 0.33 eV indicated by the membrane potential required to reverse the equilibrium I-Q ~ I Q-, which would imply that this electron transfer is exposed to 100% of the

101

membrane potential [71]. Presumably, this special role of the 1 - t o Q electron transfer is imposed by polarizability differences inside the reaction center protein and provides little information on the distance spanned. As mentioned above, the recombination of P÷Q- is largely due to a reaction which does not involve a singlet reexcitation of P. An activation enthalpy for the reaction P+Q--+ P Q of 0.33 eV [34] and of 0.15 eV [81] has been reported. The value of 0.33 eV is based on more precise data in the physiological temperature range, and would be consistent with the 0.6 eV activation enthalpy of the 3 x 104 times slower back reaction of $2 Q- [12], if the entropy difference between S1P + and S2P is small. If the activation enthalpy of the reaction P+Q--+P Q is indeed equal to the free energy difference between I-Q and I Q-, the entropy difference between I-Q and I Q- may be small (in contrast to purple bacteria [7]), since the charge recombination P+Q- ~ P Q probably proceeds via the state P+I-Q. When brought together by reversed electron transport, the electron spins on P+I- are unrelated and because there are three possible triplet configuration and only one singlet, the odds are three to one that they do not allow recombination to the singlet excited state P* (which would normally be retrapped and generate P+Q- again, anyway), Instead, a recombination to the triplet state pT cannot be avoided in this case. Although the triplet cannot be demonstrated because it decays much faster than it is formed, the triplet yield may be 97% [25] and its formation is probably the most important loss process in PS II. This loss process is inherent in the use of singlet excited chlorophyll as an energy source and should be counted among the "unavoidable losses" in thermodynamic discussions [e.g., 109]. The obvious way to minimize this loss process is to increase the energy barrier imposed by the uphill electron transfer from Q - t o I. Therefore proposals implying an apparently unnecessarily large free energy difference between P* and P+U [e.g., 94] are a priori less attractive. The situation in purple bacteria is quite different: the height of the energy barrier is not important here, because the rate of recombination by electron tunnelling limits the lifetime of P+Q- [c.f 42]. In PS II, however, this tunnelling process predominates only below about -- 70°C [81] and the energy level of P+I- is the main barrier against charge recombination at physiological temperatures. IV. The reduction of P+

When the state P+I Q- has been reached, the next reaction is the reduction of P+ by the secondary electron donor, Z. This reaction has led to much confusion in the past and it is still unclear whether there is only one Z or two different Z's-operating either in series or in parallel. This situation is largely due to the lack of methods to study Z oxidation, and to the fact that the observed P+ reduction kinetics usually contain several phases, which may range from 20 ns to a few ms.

102 The slowest phases in P÷ decay are ascribed to charge recombination with Q-. The temperature independent recombination of P÷Q-, which takes about 2ms [81], and the thermally activated 100 to 300/~s recombination at physiological temperatures (discussed above) normally prevent accumulation of the state P÷Q. Under extreme experimental conditions P+Q can be formed (e.g., at pH < 4 in the presence of ferricyanide [106], but it always oxidizes something within milliseconds. Also the oxidation of some reaction center components at low temperature, e.g., cytochrome bss9 [62] or carotenoid [89], is attributed to non-physiological electron transfer routes. A more rapid phase in P+ reduction, with a halftime of 35/~s [39], or perhaps a pair of phases with 10 and 60/Js halftimes [72], is due to charge recombination as well, but Q-is not involved here [36, 72]. This will be discussed in the last section. The slowest P÷ reduction which can be ascribed to electron donation by Z takes 2-20~s, depending on pH [14, 22], and is observed only when the oxygen evolving complex has been inactivated, e.g., by Tris-treatment. The kinetic properties of Z are drastically changed by this inactivation and for clarity the symbol Zi will be used here. On the basis of its ESR (signal IIf [11]) and optical spectrum [26, 31, 117], Z~. is thought to be a plastosemiquinone cation [29, 77], which for some mechanistic reason cannot be stabilized by deprotonation (however, see [117]) and thereby maintains a very high oxidizing potential [118]. Z~Z i equilibrates (Keq = 104) with a similar molecule, D [17]. D÷, that produces an ESR signal IIs, is amazingly stable and its function is not clear. The couple IT/D has been titrated at 0.76 V, suggesting a value of about 1.00V for Z~/Z i [18]. The back reaction of Z~-Q- is about 2 x 10 a times slower than that of P÷Q- [119], so the midpoint potential of P÷/P might be about 0.058 log (2 x 103) = 0.2 V higher than that of Z~./Zi, near 1.2 V. In the excited state (about 1.8 eV above the ground state) the midpoint potential is 1.8 V lower, so the midpoint potential of P÷/P* would be about --0.6 V, close to the value obtained by titration for I/I- [58, 86]. Titrations allow only a rough estimate of the operating potential, but apparently they are not too far off in this case. In intact oxygen evolving PS II, the kinetic behaviour of Z is complicated. The various possible kinetic schemes were evaluated more than adequately by Bouges-Bocquet [15], in the light of the evidence available in 1980. Two kineticaUy distinct electron donors, Z1 and Z2, operate in series o r - preferably - in parallel to oxidize the oxygen evolving complex, Z1 is involved in the first and the second oxidation, So -~ $1 and $1 ~ S2 in terms of Kok's S,state model [63], while the slower Z2 is involved in the third and fourth, $2 ~ $3 and $3 ~ $4. ($4 is rapidly reduced to So with concomitant release of an oxygen molecule). Z2 is probably the same component as Zi in Tris-washed PS II; the identity of Z1 is unknown. This model is still in use, only the proposed reaction rates have been corrected upwards repeatedly.

103 The most recent data on the reduction kinetics of P+ [19] indicate that Z2 is oxidized in 260 ns (halftime) and Zl in 23 ns in So and $1, and in 50 ns in $2 and Sa. The proposed equilibrium constant P Z~i/P+Z1 in 29 in So and $1, and 2.2 in $2 and $3. The S-state dependence, both of the oxidation rate of Z1 and of the associated free energy change, is attributed to the electrical charge in the oxygen evolving complex (the positive charge added in the $1 --> $2 transition, in contrast to the other transitions, is not compensated by proton release [see e.g., 37]). These data do not change the conclusions of Bouges-Bocquet qualitatively. The interpretation might apply both for a series and for a parallel arrangement of Z1 and Z2, but adds an interesting possibility to explain the effects of Tris-washing if ZI and Z2 operate in series: if Tris-treatment somehow results in an even larger electrostatic effect on the equilibrium P+Za ~ P Zi~,the equilibrium concentration of Z~"would be low, explaining its apparent absence in Tris-washed samples and explaining the slow electron transfer from Z2 to P* as well, assuming no effect of Triswashing on Z2. Tris-washing was claimed to increase the equilibrium constant P Z~/P*Z2 by at least two orders of magnitude [119], but that depends on the unknown rate constant of electron transfer from P to Z~, which remained as adjustable parameter in Bouges-Bocquet's model [15]. The model may need revision, however, to accommodate the fact that the oxidation of Z2, as confirmed by ESR measurements [13], is much faster than all S-state transitions ([28], see below). If $1 were not connected to Z2 (as suggested by the fact that it can be oxidized efficiently to $2 a t - 40°C [20], whereas Z ] is no longer formed [116] ) a small equilibrium constant of P+Z2 ~ P Z~ must indeed be assumed. Perhaps the 'electrostatic switch' for Z+I accumulation might work in the opposite direction for Z~ accumulation. Alternatively, all S-state advances may normally proceed through Z~ and some other explanation is required to account for the different temperature dependences of S~ and $2 oxidation. If Z~i turns out to be a different component, not contributing to ESR signal IIvf, this is clearly indicated by the observation that about half of signal IIvf in repetitive flashes decays with a halftime in the order of 50ps [13], corresponding with the So -+ $I and S~ ~ $2 transitions ([28], see below). It is, therefore, of special interest that two research groups recently found a donor that exhibits an ESR signal at g = 4.1, possibly due to a non-heine iron [21,122]. The signal was observed upon illumination at 200 K, and a physiological role in electron transport remains to be demonstrated. The possibility that only a single Z molecule per center exists has not yet been excluded. What is described as the reduction of Z~i by Z2 in the series model, might as well be some relaxation process in the molecular environment of Z. Now that the absorbance difference spectrum of Z i is known, the main uncertainties about this elusive part of PS II electron transport may soon be resolved. Fig. 1 summarizes the process of charge separation and stabilization in the PS II reaction center, as discussed above.

104

1.8

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E (eV) 1.6

PS II of 2931

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